Ranaviruses Evolutionary Intermediate Within Common

The Genome Sequence of the Emerging Common Midwife Toad Virus Identifies an Evolutionary Intermediate within Ranaviruses

Carla Mavian, Alberto López-Bueno, Ana Balseiro, Rosa Casais, Antonio Alcamí and Alí Alejo J. Virol. 2012, 86(7):3617. DOI: 10.1128/JVI.07108-11. Published Ahead of Print 1 February 2012. Downloaded from

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Carla Mavian,a Alberto López-Bueno,a Ana Balseiro,b Rosa Casais,b Antonio Alcamí,a and Alí Alejoc* Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spaina; Centro de Biotecnología Animal, Servicio Regional de Investigación y Desarrollo Agroalimentario, Gijón, Spainb; and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Spainc

Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic Downloaded from sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses. http://jvi.asm.org/ lobal population declines and extinction of multiple amphib- been published only for Asian, American, and Australian isolates Gian species have been reported over the last 20 years (11). As (18, 21, 24, 28, 35, 37, 41). Recently, it has been proposed that estimated by the International Union for Conservation of Nature, ranaviruses can be subdivided into two distinct groups based on the Amphibia class includes the highest number of critically en- phylogenetic analyses and genome colinearity, grouper iridovirus dangered species among the animal classes examined, with an es- (GIV)-like ranaviruses and amphibian-like ranaviruses (ALRV) timated 41% of its species under threat (23). Therefore, this class (24). The first group includes GIV and Singapore grouper irido- seems to be a particularly sensitive indicator of the current biodi- virus (SGIV), which were isolated in Asia from fish (35, 41). The versity losses associated with the global warming process and ALRVs include frog virus 3 (FV3) and Ambystoma tigrinum virus on April 5, 2012 by UNIV OF MAINE other human-related environmental factors. It has been shown (ATV), which were isolated from frogs and salamanders, respec- that habitat loss, increased human population density, and in- tively, in North America, the tiger frog virus (TFV), isolated in creased climatic variability are important factors that compromise China, the epizootic hematopoietic necrosis virus (EHNV), iso- the survival of amphibian species (34) and that multiple drivers of lated from fish in Australia, and the soft-shelled turtle iridovirus extinction are likely to coordinately accelerate amphibian declines (STIV), isolated in China (21). Within the ALRVs, the degree of in the near future (20). genome sequence colinearity is high, although two different The role of emerging infectious diseases in the rapid decline of groups of viruses can be distinguished: the EHNV/ATV group, amphibian populations is being increasingly studied. The recent which may be closer to a putative most recent common ancestor of spread of the lethal fungal pathogen Batrachochytrium dendroba- the whole group (24), and the FV3/TFV/STIV group, which shows tidis is well documented and has been associated with long-term two discrete acquired genomic inversions when compared to the amphibian population declines in several locations (6). A second genomes of the former group. widespread pathogen of amphibians that is now recognized as an Ranavirus emergence as a pathogen of amphibians and other emerging infectious disease and therefore likewise included in the ectothermic vertebrates is probably linked to their host range plas- list of notifiable diseases by the World Organization for Animal ticity as well as to environmental and ecological factors (17). The Health is ranaviruses. Although their association with population first clear evidence that viral infection can be the cause of localized declines has to be studied further (14), ranavirus infections have amphibian population declines was reported in Europe, where been clearly associated with mass mortalities of several amphibian ranaviruses have caused large-scale mortalities of the common as well as reptile and fish species worldwide (3, 36, 40). frog Rana temporaria in the United Kingdom since 1985 (40). The Ranavirus is a genus within the Iridoviridae family which in- causative agent is thought to be a variant of FV3, which may have cludes large, icosahedral viruses containing circular, double- stranded DNA genomes with sizes ranging from 105 kbp to 140 kbp and a coding potential of approximately 100 open reading Received 14 December 2011 Accepted 18 January 2012 frames (ORFs) (10). The family includes five different genera, two Published ahead of print 1 February 2012 of which infect insects while the others infect cold-blooded verte- Address correspondence to Alí Alejo, [email protected]. brates. Species from the genera Megalocytivirus and Lymphocysti- * Present address: Centro de Investigación en Sanidad Animal (INIA), Ctra. de virus have been found to infect only teleost fish, while ranaviruses Algete a El Casar, Valdeolmos, Spain. are known to infect reptiles, amphibians, and fish, and the reasons C. Mavian and A. López-Bueno contributed equally to this article. for this broad host specificity are yet unknown. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Ranavirus outbreaks have been described from different loca- doi:10.1128/JVI.07108-11 tions worldwide. However, full-length genome sequences have

0022-538X/12/$12.00 Journal of Virology p. 3617–3625 jvi.asm.org 3617 Mavian et al. been introduced through the movement of infected animals from previously annotated in other ranaviral genomes, preserving the same North America (22). number for both R and L orientations (CMTV ORFs 35 and 54). The common midwife toad virus (CMTV) was first isolated on Nucleotide-to-nucleotide comparisons between the CMTV genome and the European continental mainland in 2007 from diseased tad- other iridoviral genomes as well as among different ranaviruses were cal- poles of the common midwife toad (Alytes obstetricans) in a high- culated using the JDotter (Java Dot Plot Alignments) software with the altitude permanent water trough in the Picos de Europa National default settings. The algorithm employed by this software is described in detail in reference 9. In the dot plots generated, a straight line represents a Park in Spain (4). The virus, causing a high mortality rate in this stretch of similarity between the two sequences compared on the x and y species as well as in juvenile alpine newts in the 2008 outbreak axes. The full-length genome sequences (accession numbers) used in (Mesotriton alpestris cyreni) (5), was shown to be responsible for a the dot plot analyses were FV3 (AY548484), TFV (AF389451), ATV systemic hemorrhagic disease. Common histological findings (AY150217), EHNV (FJ433873), GIV (AY666015), SGIV (AY521625), were the presence of intracytoplasmic inclusion bodies and the and STIV (NC012637). Phylogenetic trees were constructed using necrosis of endothelial cells, the latter of which results in destruc- MEGA5 software. For the phylogenetic analysis of ranavirus MCPs, the tion of several organs, including skin, liver, and kidney. Sequence following sequences were used: ranavirus KRV-1 (KRV-1; accession no. analyses of DNA fragments belonging to the major capsid protein ADO14139), Rana catesbeiana virus-JP (RCV-JP; no. BAH80413), soft- Downloaded from (MCP) and DNA polymerase genes showed that CMTV clustered shelled turtle iridovirus (STIV; no. ABC59813), frog virus 3 (FV3; no. more closely with the ALRVs than with the GIV-like ranaviruses ACP19256), tiger frog virus (TFV; no. AAK55105), Bohle iridovirus (BIV; no. ACO90022), pike-perch iridovirus (PPIV; no. ACO90019), Rana within the Ranavirus genus. In 2010, a CMTV outbreak in a pond esculenta virus (REV; no. ACO90020), Chinese giant salamander virus in the Netherlands was described as the cause of a mass mortality (CGSV; no. ADZ47908), Ambystoma tigrinum virus (ATV; no. event affecting water frogs and common newts (29), showing that TP003785), epizootic hematopoietic necrosis virus (EHNV; no. both the host range and geographic distribution of CMTV are AAO32315), Ranavirus maxima/9995205/DNK (Rmax; no. ADI71344), much wider than previously suspected. cod iridovirus/15/04.11.92/DNK (CodV; no. ADI71343), European

The importance of ranavirus infections in amphibian popula- sheatfish virus (ESV; no. ACO90018), European catfish virus (ECV; no. http://jvi.asm.org/ tion declines as well as the lack of knowledge about the nature of ACO90017), short-finned eel ranavirus (SERV; no. ACO90021), large- circulating European ranaviruses prompted us to determine the mouth bass ulcerative syndrome virus (LMBUSV; no. ADB77863), large- complete genome sequence of the emerging CMTV. mouth bass virus (LMBV; no. CBW46836), grouper iridovirus (GIV; no. AEI85923), Singapore grouper iridovirus (SGIV; no. AAS18087), king grouper iridovirus (KGIV; no. AEI85924), crimson snapper iridovirus MATERIALS AND METHODS (CSIV; no. AEI85915), and lymphocystis disease virus from China Virus and cells. CMTV isolated from alpine newts (Mesotriton alpestris (LCDV-C; no. YP_025102). cyreni) without plaque purification (4) was amplified in a single step on ze- Nucleotide sequence accession number. The genome sequence of brafish ZF4 cells (ATCC CRL-2050) at 28°C in Dutch-modified RPMI me- CMTV was deposited into GenBank under accession no. JQ231222. on April 5, 2012 by UNIV OF MAINE dium containing 2% fetal calf serum. Supernatants from CMTV-infected ZF4 cells were collected, and viral particles were obtained by ultracentrifugation at RESULTS ϫ 20,000 g through a 36% sucrose cushion for1hat4°C. CMTV is a distinct ranavirus isolated in Europe. To better un- Isolation of viral DNA. The virus particles were treated with DNase I derstand the taxonomic position of the CMTV, we sequenced its and S7 micrococcal nuclease to digest free DNA. After proteinase K treat- MCP gene and performed a phylogenetic analysis comparing the ment, viral DNA was extracted with phenol-chloroform and precipitated with sodium acetate and ethanol in the presence of 10 ␮g of glycogen MCP gene to that of 23 other ranaviruses. As shown in Fig. 1A, the (Roche) as the carrier. Extracted DNA was randomly amplified using CMTV clustered very closely with the Chinese giant salamander Phi29 DNA polymerase (GenomiPhi V2; GE Healthcare). virus, isolated in China in 2010 (16), and the Rana esculenta virus, Sequencing and assembly of the viral genome. Amplified viral DNA isolated from edible frogs (Pelophylax esculentus) in Italy and Den- was pyrosequenced on a 454 GS FLX instrument housed in the Parque mark in 2009 (3). Together with the pike-perch iridovirus, iso- Científico de Madrid. The output consists of 40,271 sequences with an lated from pike-perch fry in Finland in 1998 (38), all four viruses average size of 375 bp. Reads were assembled in two steps with Newbler formed a distinct group that was included within the previously 2.5.3 (Roche-454 Life Science). First, a de novo assembly under stringent described ALRVs but clearly separated from its two proposed ma- parameters (97% minimum overlap identity in at least 250 bp) generated jor branches, represented by FV3 and EHNV. Collectively, the 2 contigs flanked by repeated sequences. Then a unique contig was ob- CMTV-like viruses are found to be more closely related to the FV3 tained by aligning the reads to the de novo genome under more relaxed parameters (98% identity in at least 50 bp). PCR amplification and Sanger group, suggesting both may have diverged from a common ances- sequencing were carried out in order to define homopolymer sequencing tor. Finally, the CMTV was distantly related to other ranaviruses, ambiguities at positions 73,736 and 3,392 and a polymorphism at position such as Ranavirus maxima/9995205/DNK, cod iridovirus/15/ 11,812 as well as the precise number of TGTGAAGCGTAAGTCCCC re- 04.11.92/DNK (2), European sheatfish virus (1), and European peats at position 66,494. The number of repetitions of the microsatellite catfish virus (32), that were isolated from fish on the European region detected at position 38,092 was determined by running a specific continent. As no full-length genome sequences for ranaviruses PCR product in a 4% agarose gel (see Fig. 2). Sequences of primers are isolated in Europe have been described to date (Fig. 1B), and given available upon request. the apparently distinct position of CMTV within the ALRVs, we Genome analysis and annotation. Open reading frames (ORFs) were decided to sequence its complete genome. numbered consecutively from the same arbitrary start point as in ATV CMTV is a typical ALRV. The complete genomic sequence of and EHNV (24, 28), and transcriptional sense was indicated by R (right) or L (left). The genome was annotated with the genome annotation trans- the CMTV was obtained using pyrosequencing combined with fer utility (GATU) software (39) using the genome of STIV as a template Sanger sequencing of PCR products for unresolved regions. After and further refined manually by using the similarity search algorithm assembly, we obtained a final genome size of 106,878 bp, with an BLASTP (http://blast.ncbi.nlm.nih.gov/) on all unassigned ORFs longer average coverage of 128 reads per position. The genome size of than 120 bp. Overlapping ORFs were annotated only if they had been CMTV is smaller than that of the EHNV (127,011 bp) and slightly

3618 jvi.asm.org Journal of Virology Genome Sequence of the Common Midwife Toad Virus Downloaded from http://jvi.asm.org/ on April 5, 2012 by UNIV OF MAINE

FIG 1 CMTV is a novel amphibian-like ranavirus isolated in Europe. (A) Phylogenetic tree of ranavirus major capsid protein sequences. The sequences were aligned with ClustalW and trimmed manually to the 450-amino-acid sequence available for the Chinese giant salamander virus (CGSV) MCP, and phylogenetic reconstruction was obtained by neighbor-joining with 1,000 bootstrap replicates. The consensus bootstrap tree is shown, with confidence values indicated on the branches. Branches with bootstrap values below 50% are collapsed. Lymphocystis disease virus from China (LCDV-C) was used as the outgroup. Viruses infecting amphibian or reptile hosts are marked with an asterisk. The GenBank accession numbers of the Iridovirus MCP sequences used for this analysis are listed in Materials and Methods. (B) World map showing the locations of isolation and the hosts of the published full-length ranavirus genome sequences and CMTV. larger than those of the other fully sequenced ALRVs ATV (106,332 bp), FV3 (105,903 bp), STIV (105,890 bp), and TFV (105,057 bp). The 55.3% GC content of the CMTV genome is similar to those of the other ALRVs, which range from 54% to 55%, but higher than those of the GIV (49%) and SGIV (48%) ranaviruses. The microsatellite identified in FV3 (12) and STIV was also found in CMTV. The use of primers specific for the flanking se- quences produced a larger PCR amplification product on CMTV DNA than on FV3 DNA, showing that the region in CMTV con- tains 60 dinucleotide repeats, compared to the 34 copies observed in the other two viruses, and that it can therefore be used for differentiation of these viruses (Fig. 2). FIG 2 PCR amplification of the microsatellites in CMTV and FV3. The mic- rosatellites of FV3 (lane 2) and CMTV (lane 3) were PCR amplified using The CMTV genome was found to contain 104 ORFs (Fig. 3) specific primers and run on a 4% agarose gel together with reference PCR encoding putatively expressed proteins with predicted molecular products of known sizes (lane 4, 170 bp; lane 5, 174 bp; lane 6, 184 bp). Lanes masses ranging from 5.2 kDa to 144.2 kDa and with conserved 1 and 7, molecular weight markers.

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FIG 3 Linear schematic organization of CMTV genome. Predicted open reading frames (ORFs) are represented as arrows indicating the size and direction of transcription. The black lines represent the CMTV genome and are divided into 30-kb segments. ORFs in the forward strand are drawn above the genome line and those in the complement strand are drawn below. Iridovirus core genes, ranavirus-specific genes, and amphibian-like ranavirus-specific genes are indicated in red, green, and blue, respectively. The asterisk shows the position of the microsatellite repetition.

domains assigned to structural proteins as well as proteins poten- nated protein sequences derived from the 26 iridoviral core genes on April 5, 2012 by UNIV OF MAINE tially involved in replication, transcription, and host response (15). As previously reported, the Asian fish viruses GIV and SGIV modification (Table 1). For 101 CMTV-predicted proteins, ortho- formed a separated branch within the fully sequenced ranaviruses logues were readily found in other ranavirus (24) genomes, with (Fig. 4). CMTV was found to cluster very confidently within the identities ranging from 65% to 100%. Only three ORFs (11L, 96L, FV3-like group, which was clearly resolved from the EHNV-like and 100L) had not been previously annotated in other ranavi- group. Furthermore, CMTV sequences were found to have a ruses, but no significant similarities to other known proteins were slightly higher similarity to the FV3/STIV group than to TFV. found in the databases. Early stop codons or frameshift mutations To determine the degree of colinearity of the CMTV genome account for the absence of annotated protein orthologues for with those of other ranaviruses, we performed dot plot analyses these three ORFs in other ALRVs in spite of their relatively high (Fig. 5 and data not shown). As expected, CMTV did not show conservation at the nucleotide level. Additionally, a genomic in- major colinearity stretches when compared to the GIV-like fish version in the FV3 group split the CMTV ORF 100L in these three ranaviruses (data not shown). As described before, ALRVs can be ranaviruses. Whether these proteins are truly expressed and func- divided into two separate groups in which colinearity is main- tional during CMTV infection remains to be addressed. tained, one including EHNV and ATV and the second including Analyses of all sequenced iridovirus genomes have identified FV3, STIV, and TFV. The CMTV genome was found to not be 26 conserved genes, which make up the core iridovirus genes (15). colinear with either of these groups. Specifically, compared to As expected, these genes are conserved in CMTV. Additionally, 27 EHNV, an inversion of the CMTV genomic segment located be- more genes were described to be conserved throughout the Rana- tween positions ϳ62,000 and ϳ91,000 was observed, while in virus genus based on the EHNV gene content (24). These include comparison to FV3, a single different inversion affecting positions a multigene family with five members in EHNV of which only two ϳ15,000 and ϳ105,000 was detected. These sites of genomic re- are retained in ATV and the FV3 group as well as in CMTV (ORFs arrangements correspond exactly to those of the double genomic 82L and 83L). Finally, the set of 13 genes conserved in all ALRVs inversion identified previously when FV3 was compared to EHNV (24) was also found in CMTV. Altogether, these results show that (24). This suggests that CMTV or its ancestor may occupy an the CMTV can be classified as a typical ALRV in terms of gene intermediate position in the evolutionary process that gave rise to content. the FV3/STIV/TFV group from the EHNV-like ancestor. Thus, CMTV represents an intermediate in ALRV evolution. Due inversion of one segment from an EHNV-like precursor may have to the Ͼ90% identity among ranavirus MCP sequences, phyloge- produced a CMTV ancestor. A further inversion might have then netic studies based exclusively on these sequences may be insuffi- produced the genomic structure found in the FV3/TFV group. cient for differentiating among ranavirus types. To further estab- Upon closer inspection of the corresponding dot plots, CMTV lish the phylogenetic relationships between CMTV and other fully was found to contain no major deletions and only three DNA sequenced ranaviruses, we performed an analysis of the concate- sequence insertions of about 500 bp each compared to FV3 (Fig.

3620 jvi.asm.org Journal of Virology Genome Sequence of the Common Midwife Toad Virus

TABLE 1 Predicted CMTV open reading framesa EHNV FV3 Best BLASTP hitc orthologued orthologued Predicted function and conserved ORF Position (nt range) Product size (aa) domain or signatureb ORF % ID Accession no. ORF % ID ORF % ID 1R 16–786 256 Putative replication factor; FV3 1R 98 YP_031579 100R 98 1R 98 pfam04947 2L 1,497–2,510 337 Putative myristylated membrane STIV 2L 90 YP_002854233 1L 91 2L 90 protein 3L 2,548–3,387 279 EHNV 2L 97 ACO25192 2L 97 NA 4R 3,419–4,633 404 TFV 4R 98 ABB92272.1 3R 97 3R 96 5R 4,674–4,856 60 FV3 4R 97 YP_031582.1 4R 92 4R 97 6R 5,291–5,893 200 cl03568 FV3 5R 86 YP_031583.1| NA NA 5R 86 7L 6,823–7,251 142 STIV 9L 95 YP_002854240.1 6L 78 NA

8R 7,246–11,211 1,321 Largest subunit of DNA-dependent TFV 8R 99 AAL77794.1 7R 98 8R 98 Downloaded from RNA polymerase; cl11429 9L 11,566–14,412 948 Helicase; smart00487 FV3 9L 98 YP_031587.1 8L 98 9L 98 10R 14,428–14,841 137 ATV p8 99 YP_003779.1 9R 97 10R 96 11L 15,222–15,455 77 NA NA 12L 15,499–16,593 364 Putative DNA repair protein; Rana grylio virus 98 AAY43134.1 10L 97 95R 98 cl14812, cl14815 9808 XPG 13R 16,689–17,156 155 pfam10881 FV3 94L 97 YP_031673.1 11R 97 94L 97 14R 17,217–17,432 71 STIV 98L 85 YP_002854329.1 12R 95 93L 96

15L 18,167–19,354 395 Immediate early protein ICP-46 STIV 97R 99 YP_002854328.1 13L 98 91R 98 http://jvi.asm.org/ 16L 19,478–20,869 463 Major capsid protein; pfam04451 PPIV MCP 99 ACO90019.1 14L 99 90R 98 17L 20,962–22,041 359 STIV 95R 88 YP_002854326.1 15L 88 89R 86 18L 22,109–22,561 150 Thiol oxidoreductase; pfam04777 STIV 94R 100 YP_002854325.1 16L 97 88R 99 19R 22,594–24,393 599 TFV 93L 95 ABB92341.1 17R 94 87L 96 20R 24,746–24,961 71 TFV 92L 80 ABB92340.1 NA 86L 75 21L 25,406–25,993 195 Thymidine kinase; cd01673 TFV 91R 98 ABB92339.1 18L 96 85R 96 22L 26,068–26,805 245 Proliferating cell nuclear antigen EHNV 19L 99 ACO25209.1 19L 99 84R 98 23L 27,222–27,866 214 Cytosine DNA methyltransferase; FV3 83R 99 YP_031662.1 20L 99 83R 99 on April 5, 2012 by UNIV OF MAINE cl12011 24L 28,233–28,520 95 Thymidylate synthase; cl00358 EHNV 21L 94 ACO25211.1 21L 94 NA 25L 28,816–29,289 157 Putative immediate early protein; EHNV 22L 97 ACO25212.1| 22L 97 82R 93 cl12687 26L 29,418–29,696 92 Transcription elongation factor SII; FV3 81R 97 YP_031660.1 23L 97 81R 97 cl02609 27R 29,752–30,870 372 RNase III; cd00593, TIGR02191 EHNV 24R 99 ACO25214.1 24R 99 80L 99 28L 31,495–33,213 572 STIV 86R 95 YP_002854317.1 25L 83 79R 95 29R 33,298–33,987 229 STIV 85L 94 YP_002854316.1 26R 95 78L 95 30R 34,687–35,034 115 TFV 82L 99 ABB92335.1 27R 96 77L 98 31L 35,031–35,252 73 TFV 81R 99 ABB92334.1 28L 96 76R 96 32R 35,315–35,569 84 LITAF-like protein; cl02754 FV3 75L 100 YP_031654.1 29R 99 75L 100 33R 35,626–36,807 393 EHNV 30R 97 ACO25220.1 30R 97 74L 85 34R 36,947–37,999 350 Putative NTPase/helicase EHNV 31R 98 ACO25221.1 31R 98 73L 97 35R 38,496–39,212 238 TFV 77L 98 ABB92331.1 32R 100 72L 97 35L 38,502–38,984 160 EHNV 33L 96 ACO25223.1 33L 96 NA 36L 39,269–39,502 77 FV3 71R 96 YP_031650.1 34L 90 71R 96 37L 39,542–39,916 124 FV3 70R 98 YP_031649.1 35L 97 70R 98 38L 39,934–40,200 88 FV3 69R 100 YP_031648.1 36L 99 69R 100 39R 40,318–40,782 154 cl00060 EHNV 37R 97 ACO25227.1 37R 97 NA 40R 41,407–42,570 387 Ribonucleotide reductase, small ATV p39 99 YP_003810.1 38R 98 67L 98 subunit; cd01049 41R 42,625–42,978 117 STIV 70L 97 YP_002854301.1 39R 90 66L 91 42R 43,115–43,459 114 EHNV 40R 89 ACO25230.1 40R 89 65L 96 43L 43,852–44,139 95 CARD-like caspase; cl14633 STIV 67R 97 YP_002854298.1 41L 96 64R 93 44L 44,250–44,744 164 dUTPase; cd07557 STIV 66R 98 YP_002854297.1 42L 97 63R 96 45L 44,861–45,403 180 STIV 65R 92 YP_002854296.1 NA NA 46R 45,123–48,776 1,217 DNA-dependent RNA polymerase FV3 62L 98 YP_031641.1 43R 96 62L 98 B subunit; cd00653, cl04593, COG0085 (Continued on following page)

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TABLE 1 (Continued) EHNV FV3 Best BLASTP hitc orthologued orthologued Predicted function and conserved ORF Position (nt range) Product size (aa) domain or signatureb ORF % ID Accession no. ORF % ID ORF % ID 47L 49,315–52,356 1,013 DNA polymerase; cl10023, cl10012 STIV 63R 99 YP_002854294.1 44L 99 60R 99 48R 52,518–53,576 352 FV3 59L 98 YP_031638.1 45R 97 59L 98 49L 54,062–54,616 184 EHNV 46L 98 ACO25236.1 46L 98 NA 50L 54,667–54,912 81 cl08321 EHNV 47L 88 ACO25237.1 47L 88 NA 51L 54,995–56,491 498 Putative phosphotransferase STIV 60R 99 YP_002854291.1 48L 98 57R 98 52L 56,532–56,936 134 EHNV 49L 98 ACO25239.1 49L 98 NA 53R 56,973–57,122 49 EHNV 50R 100 ACO25240.1 50R 100 NA 54R 57,130–58,425 431 Helicase; cd00046, COG1061 TFV 56L 98 AAL77803.1 51R 98 55L 97 54L 57,142–58,281 379 FV3 55R 97 YP_031634.1 p40 97 55R 97

55R 58,338–58,730 130 STIV 56L 98 YP_002854287.1 NA NA Downloaded from 56L 58,875–60,443 522 Myristylated membrane protein; FV3 53R 99 YP_031631.1 53L 98 53R 99 pfam02442 57R 60,780–61,847 355 3-beta-hydroxysteroid STIV 54L 99 YP_002854285.1 54R 97 52L 98 dehydrogenase; pfam01073 58L 62,104–63,789 561 TFV 53R 98 ABB92313.1 84R 98 51R 98 59R 63,868–65,379 503 cl02640, PTZ00108 STIV 52L 96 YP_002854283.1 83L 97 49L 98 60R 65,427–65,738 103 ATV p78 88 YP_003849.1 82L 98 48L 98 61R 65,741–66,157 138 TFV 49L 99 ABB92309.1 81L 96 47L 98

62R 66,282–66,749 155 Neurofilament triplet H1-like STIV 49L 94 YP_002854280.1 80L 83 46L 83 http://jvi.asm.org/ protein; PTZ00449 63R 66,859–67,269 136 FV3 45L 98 YP_031623.1 79L 98 45L 98 64R 67,396–68,364 322 TFV 46L 80 ABB92307.1 78L 96 42L 98 65L 68,753–72,331 1,192 FV3 41R 98 YP_031619.1 77R 98 41R 98 66L 72,321–72,461 46 ATV p71 91 YP_003842.1 76R 70 NA 67L 72,647–73,330 227 ATV p70 84 YP_003841.1 75R 80 40R 79 68L 73,346–73,696 116 EHNV 74R 97 ACO25264.1 74R 97 39R 94 69L 73,805–75,502 565 Ribonucleoside-diphosphate TFV 41R 99 AAL77800.1 73R 99 38R 98 on April 5, 2012 by UNIV OF MAINE reductase, large subunit; cd01679, PRK09102 70L 75,641–76,276 211 Putative NIF/NLI interacting EHNV 72R 99 ACO25262.1 72R 99 37R 98 factor; cl11391 71R 76,657–76,956 99 FV3 36L 89 YP_031614.1 NA 36L 89 72R 77,010–77,228 72 FV3 36L 93 YP_031614.1 NA 36L 93 73R 77,268–77,846 192 EHNV 71L 65 ACO25261.1 71L 65 35L 64 74L 77,831–78,151 106 EHNV 70R 94 ACO25260.1 70R 94 34R 95 75L 78,293–78,484 63 EHNV 69R 97 ACO25259.1 69R 97 33R 97 76L 78,567–80,717 716 Neurofilament triplet H1-like STIV 35R 84 YP_002854266.1 68R 85 32R 81 protein; cl06505, cl06430 77L 80,767–81,186 139 cl10444 EHNV 67R 98 ACO25257.1 67R 98 31R 95 78L 81,674–81,940 88 EHNV 66R 100 ACO25256.1 66R 100 NA 79L 82,077–82,565 162 STIV 32R 99 YP_002854263.1 63R 92 28R 98 80L 82,614–85,544 976 Putative tyrosine kinase; cl14933 STIV 31R 98 YP_002854262.1 62R 96 27R 97 81L 86,043–86,855 270 eIF2a-like protein; cl09927 EHNV 61R 95 ACO25251.1 61R 95 26R 83 82L 87,180–88,094 304 p31K protein; cl12506 EHNV 60R 97 ACO25250.1 60R 97 25R 99 83L 88,158–89,255 365 FV3 24R 98 YP_031602.1 57R 96 24R 98 84L 89,680–90,828 382 FV3 23R 98 YP_031601.1 56R 97 23R 98 85L 91,206–94,133 975 Putative D5 family NTP/ATPase; STIV 25R 99 YP_002854256.1 85L 98 22R 99 cl11759, cl07361, cl07360 86R 94,258–94,926 222 ATV p82 95 YP_003853.1 86R 95 21L 95 87L 95,163–95,609 148 STIV 23R 99 YP_002854254.1 88L 98 20R 96 88L 95,657–98,443 928 cl07414 TFV 19R 96 ABB92284.1 89L 88 19R 94 89R 98,232–98,468 78 FV3 18L 97 YP_031596.1 NA 18L 97 90R 98,505–100,013 502 STIV 18L 99 YP_002854249.1 90R 99 17L 99 91L 99,224–99,730 168 STIV 19R 99 YP_002854250.1 NA NA 92R 100,050–100,997 315 STIV 17L 97 YP_002854248.1 91R 98 NA 93L 100,254–101,081 275 Putative integrase homologue FV3 16R 99 YP_031594.1 NA 16R 99 94L 101,361–102,308 315 Putative A32-like virion packaging STIV 16R 99 YP_002854247.1 92L 97 15R 97 ATPase; cl04659, smart00382 (Continued on following page)

3622 jvi.asm.org Journal of Virology Genome Sequence of the Common Midwife Toad Virus

TABLE 1 (Continued) EHNV FV3 Best BLASTP hitc orthologued orthologued Predicted function and conserved ORF Position (nt range) Product size (aa) domain or signatureb ORF % ID Accession no. ORF % ID ORF % ID 95L 102,405–102,764 119 STIV 15R 100 YP_002854246.1 93L 98 14R 100 96L 102,841–103,035 64 NA NA 97R 103,076–103,270 64 TFV 13L ABB92279.1 NA NA 98R 103,382–104,425 347 STIV 14L 98 YP_002854245.1 95R 97 12L 98 99L 104,491–104,703 70 STIV 13R 99 YP_002854244.1 96L 99 11R 97 100L 104,769–104,990 73 NA NA 101R 105,187–105,873 228 EHNV 98R 97 ACO25288.1 98R 97 96R 94 102R 105,939–106,352 137 STIV 103R 99 YP_002854334.1 99R 96 97R 99 a nt, nucleotides; aa, amino acids; ID, identity. b Predicted function is based on conserved domains and/or previous annotation in other ranaviruses. LITAF, lipopolysaccharide-induced tumor necrosis factor alpha (TNF-␣). Downloaded from c Significant hits using conserved domain search at NCBI BLASTP are shown. d NA, not applicable (BLASTP E value Ͼ 0.001).

5). These insertions corresponded to sequences that are also found represent, in terms of gene content and genomic structure, a very in EHNV and to different degrees in ATV and TFV but that are recent ancestor of the previously described group of FV3-like vi- specifically lost in FV3 and STIV. In particular, the insertions af- ruses (24). As the CMTV forms a cluster with ranaviruses isolated fect CMTV 24L, CMTV 39R, and CMTV 81L, which encode a from different species and locations worldwide, including fish and truncated thymidylate synthase protein, a putatively secreted pro- amphibian viruses, we believe that these may form a single, novel http://jvi.asm.org/ tein containing an intact FGF domain, and an eIF2␣-like protein, group of ALRVs. The information provided by this report should respectively. Conversely, compared to EHNV, CMTV presents at therefore be useful to specifically address the relationship of these least 14 deletions and two insertions, which affect CMTV 6R as viruses with CMTV and to further define the taxonomic positions well as CMTV 71R and CMTV 72R, that are also found in TFV, of different ranavirus isolates, which have been poorly described FV3, and STIV. This pattern of sequence gain and loss among at the genetic level. ALRVs is also consistent with the intermediate position of CMTV One of the most outstanding features of ranaviruses is their

in the evolution of EHNV-like viruses toward FV3-like viruses, wide host range, which includes anuran and urodele amphibians, on April 5, 2012 by UNIV OF MAINE since CMTV retains EHNV-like characteristics which are lost in reptiles, and teleost fish. It has been proposed that the most recent FV3-like viruses and has already acquired sequences found previ- common ancestor of all ALRVs was most probably a fish virus, ously only in FV3-like viruses. Therefore, the genomic content with independent events giving rise to isolates infecting different and structure of CMTV may resemble those of the last common hosts (24). Experimental evidence suggests that ATV may infect ancestor of TFV, STIV, and FV3 viruses. only urodele hosts, while neither fish nor frogs are susceptible to ATV infection (25). As CMTV can infect both anuran and urodele DISCUSSION amphibians in the wild (4, 5, 29), it is tempting to speculate that In this report, we have shown that the CMTV, the first European CMTV may have derived from an ATV-related virus, acquiring ranavirus to be completely sequenced, should be classified as an the ability to infect frogs. Potentially, further divergence into the ALRV. Based on our analyses, we propose that the CMTV might FV3-like viruses may have produced frog- and reptile-specific vi- ruses. However, FV3 infections of both fish and salamander spe- cies have been reported (30), and the Bohle iridovirus, isolated from the ornate burrowing frog in Australia, is also known to infect fish (31). Therefore, both data from experimental infections and a clearer taxonomic description of ranaviruses based on com- plete genome information will help address this issue. As described above, CMTV has retained some features from the EHNV-like group which are lost in FV3-like viruses and has acquired potential coding sequences which were previously identified only in the FV3-like viruses, further supporting their intermediate position in the evolution between these groups of ALRVs. Interestingly, some of these coding regions may play a role in host range or virulence. In particular, a potentially functional protein belonging to the fibro- blast growth factor family is present only in EHNV, ATV, and CMTV. In baculoviruses, such an activity was shown to be able to FIG 4 CMTV is closely related to the FV3-like viruses. Phylogenetic analysis of induce cell migration, suggesting a role in immunomodulation or the concatenated sequences of the 26 iridovirus core proteins (15) from the viral spread (13). Additionally, the eIF2␣-like protein (vIF2␣H) indicated viruses. The sequences were aligned with ClustalW, and the phylo- genetic reconstruction was obtained by neighbor-joining with 1,000 bootstrap present in most ALRVs, including CMTV, but truncated in the replicates. Consensus bootstrap confidence values are indicated above the FV3 and STIV has been demonstrated to act as an inhibitor of the branches. LCDV-C was used as the outgroup. antiviral protein kinase PKR (33). Thus, ATV mutants lacking

April 2012 Volume 86 Number 7 jvi.asm.org 3623 Mavian et al. Downloaded from

FIG 5 CMTV is an evolutionary intermediate in ALRVs. Dot plot comparisons of the CMTV genome and the EHNV and FV3 genomes. For clarity, the reverse complement sequence of the FV3 genome was used in these comparisons. As a reference, a dot plot of EHNV versus FV3 is shown in the right panel. Black arrows indicate the sites of inversion in the CMTV genome compared to the FV3 genome, and gray arrows indicate the sites of inversion in the CMTV genome compared http://jvi.asm.org/ to the EHNV genome. Triangles indicate sequences present in CMTV but absent in FV3 or EHNV. vIF2␣H activity were shown to be more sensitive to the antiviral lands, animals affected with CMTV showed no evidence of Batra- activity of interferon (IFN) and displayed an increased time to chochytrium dendrobatidis infection (29), suggesting that the two death in infected salamanders, demonstrating the protein’s role as pathogens did not occur simultaneously. Whether an interaction be- an important virulence factor in vivo (27). Conversely, CMTV 6R, tween the pathogens is important for amphibian extinction dynamics which is found in ALRVs except EHNV and ATV, shows signifi- remains to be addressed. Mass mortality events affecting the urodele on April 5, 2012 by UNIV OF MAINE cant similarity to DNA and protein sequences from frog species, Triturus marmoratus in northwest Portugal in 2003 are probably also suggesting a recent acquisition event during the process of adap- related to ranavirus outbreaks, and recently, a novel ranavirus closely tation to novel amphibian hosts. Implications of these specific related to FV3 was isolated from lizards in Portugal (12). The nature events of sequence gain and loss among ALRV groups on virus of these viruses and whether they may represent different isolates of host range or pathogenicity remain to be addressed. the CMTV are currently unknown. Although the distribution and Whether the broad host range of ranaviruses, compared to that of degree of prevalence of CMTV in different host species need to be other double-stranded DNA viruses like orthopoxviruses, is an inher- studied in greater detail, the presence of CMTV in the Netherlands ent feature of the iridoviruses or related to a more recent evolutionary (29) suggests that this virus may show a wide geographic distribution history of this virus family is an important topic for study. While gene across the European mainland. In the case of tiger salamander infec- loss has been established as one of the main driving forces in poxvirus tions in North America, it was shown that while ATV has coevolved evolution (19), we observe very little differences in terms of global with its host in their geographic range, human-mediated transfer of gene content among ALRVs, which might indicate that this group of ATV-infected bait salamanders introduced novel, more virulent viruses is only on the verge of an evolutionary radiation. This may strains to discrete locations, resulting in disease emergence (26, 36). It relate to the human-mediated exchange of host species or viruses will be important to determine the mechanisms of emergence of between distant regions, which increases the number of potential CMTV-caused disease and whether it may be linked to environmen- hosts to infect and is consistent with their established character as tal factors, virus spread, or both. emerging infectious-disease-causing agents. As advances in sequencing technologies make more genomic As pathogens of wildlife, ranaviruses have been found to cause information on the ranavirus isolates available, a better under- mass mortalities mainly in amphibian populations worldwide. One standing of the evolutionary history of this virus family will be of the earliest amphibian population declines to be reported was that affecting the common midwife toad and other amphibian species, gained. In particular, a better knowledge of the ranavirus species including salamanders, during the last decade in Spain (7, 8). These and strain structure will be crucial to set up detection methods and declines have been associated with the emergence of chytridiomyco- surveillance strategies in order to minimize their impact on am- sis, a disease caused by the fungal pathogen Batrachochytrium dendro- phibian wildlife and cultivated species. batidis. Recent studies have shown the widespread yet heterogeneous distribution of Batrachochytrium dendrobatidis across the Iberian ACKNOWLEDGMENTS Peninsula and predict further spread of the disease in the future (42). This work was supported by grant AGL 2009-08711 from the Spanish However, the isolation of CMTV from diseased amphibians in Spain Ministerio de Ciencia e Innovación. Alberto López-Bueno and Carla Ma- suggests that ranavirus infection, too, may be having an impact on vián are recipients of the Ramón y Cajal and Formación del Personal population declines, as reported for other locations. In the Nether- Investigador fellowships, respectively, from the same institution.

3624 jvi.asm.org Journal of Virology Genome Sequence of the Common Midwife Toad Virus

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